Tannerella forsythia is major periodontal pathogen implicated in the development and severity of periodontal disease pathogenesis. In spite of its importance in periodontitis T. forsythia remains one of the most understudied periodontal pathogens, partly due to its fastidious and slow growth as well as of the lack of good genetic system useful for manipulation of this organism. So far, only a few virulence factors have been described for T. forsythia. It has been showed its interaction with epithelial cells and synergisti relations with other bacteria in the oral community such as Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola. Although not always possible, the best way to study a microorganism is within its own habitat, in situ. We characterized in vivo gene expression profiles of subgingival biofilm samples in advanced periodontitis using metatranscriptomic analyses. A comparison of data from periodontitis and health revealed the presence of a highly up-regulated region in the genome of T. forsythia during periodontal disease. Interestingly, after hits normalization this region was highly up regulated. We believe that the high expression of this region during disease may indicate its clinical relevance. Two different annotations have been proposed for the transcripts by the Human Oral Microbiome Database (HOMD): a cell wall hydrolase and a Dipeptidyl Peptidase IV. The principal Aim for this proposal is: To characterize the most highly expressed proteins of T. forsythia during periodontal disease. The first step will be elucidating whether that region encodes one or two proteins. In the second part of the project the protein will be characterized in order to identify ts enzymatic activity and clarify which of the two annotations by HOMD is correct. T. forsythia requires external sources of N-acetylmuramic acid to grow. It has been hypothesized that T. forsythia may assist dampening of inflammation and evading the host immune system through the degradation of peptidoglycan released from the oral biofilm which is toxic for the host. In case we detect cell wall hydrolase activity we will pursue that hypothesis. If DDPIV protease activity is identified, T. forsythia may be directly involved in host tissue destruction and we woud have identified a new virulence factor. The studies proposed in this application are aimed at understanding the functions of highly up-regulated T. forsythia proteins during in periodontal disease.